The fermentation method that is a method of producing a substance, which involves culturing a microorganism or cultured cells, can be roughly classified into (1) a batch fermentation method or a fed-batch fermentation method and (2) a continuous fermentation method.
The batch or fed-batch fermentation method has advantages such as simple facilities and less damage by contaminating bacteria because it is short term cultivation. However, the concentration of a product in a culture is increased with time to reduce productivity and yield due to the influence of osmotic pressure or inhibition by the product. Accordingly, it is difficult to maintain high yield and high productivity stably for a long time.
The continuous fermentation method is characterized in that high yield and high productivity can be maintained for a long time by preventing accumulation of an objective substance at high concentration in a fermenter. Continuous fermentation methods for fermentation of L-glutamic acid and L-lysine have been disclosed (Non-Patent Document 1). In these examples, however, raw materials are continuously fed to a culture while a culture containing microorganisms and cells is withdrawn, so that the microorganisms and cells in the culture are diluted, and therefore the improvement in production efficiency is limited.
In the continuous fermentation method, it has been proposed that microorganisms and cultured cells are filtered with a separation membrane to recover a product from a filtrate, while the filtered microorganisms and cultured cells are retained in, or refluxed to, a culture thereby maintaining a high density of the microorganisms and cultured cells in the culture.
For example, techniques of continuous fermentation in a continuous fermentation apparatus using a ceramics membrane have been disclosed (Patent Documents 1, 2 and 3). However, the disclosed techniques has a problem in the reduction in filtration flow rate and filtration efficiency caused by clogging of the ceramics membrane, and for prevention of this clogging, reverse washing or the like conducted.
A process for producing succinic acid by using a separation membrane has been disclosed (Patent Document 4). In this technique, a high filtration pressure (about 200 kPa) is used in membrane separation. This high filtration pressure not only has disadvantage in costs but also causes physical damage to microorganisms and cells by pressure in filtration treatment and is thus not suitable for continuous fermentation wherein microorganisms and cells are continuously returned to a culture.
Conventional continuous culture is a culture method wherein a fresh medium is fed at a constant rate to a fermenter, and a culture in the same amount as the medium is discharged from the fermenter, thereby keeping the fluid volume in the fermenter always constant. In batch culture, culture is finished when an initial substrate is consumed, whereas in continuous culture, culture can be theoretically continued infinitely. That is, infinite fermentation is theoretically feasible.
In the conventional continuous culture, on the other hand, microorganisms together with a culture are discharged from a fermenter so that the density of microorganisms in the fermenter is hardly kept high. If fermenting microorganisms can be kept at high density in fermentation production, the efficiency of fermentation production per fermentation volume can be improved. For this purpose, microorganisms should be retained in, or refluxed to, a fermenter. The method wherein microorganisms are retained in, or refluxed to, a fermenter includes a method that involves solid-liquid separation of a discharged culture by gravity, for example centrifugation, and returning precipitated microorganisms to a fermenter, and a method that involves filtration to separate microorganisms as solids and discharging a culture supernatant only from a fermenter. However, the method using centrifugation is not practical because of high power cost. The method using filtration requires high pressure for filtration as described above and has been examined mainly at the laboratory level.
As described above, the conventional continuous fermentation methods suffer from various problems and are hardly industrially applicable.
That is, it is still difficult in the continuous fermentation method to achieve high substance productivity by filtering microorganisms and cells with a separation membrane thereby recovering a product from a filtrate and simultaneously refluxing the filtered microorganisms and cells to a culture to improve the density of the microorganisms and cells in the culture and to keep their density high. Hence, there have been demands for innovations in techniques.
Patent Document 1: Japanese Patent Application Laid-open (JP-A) No. 5-95778
Patent Document 2: JP-A No. 62-138184
Patent Document 3: JP-A No. 10-174594
Patent Document 4: JP-A No. 2005-333886
Non-Patent Document 1: Toshihiko Hirao et. al., Appl. Microbiol. Biotechnol., 32, 269-273 (1989)